The processes of transcription and translation are fairly well understood, but only recently have the factors which determine the final intra- or intercellular location of a protein been investigated. This project proposes to define the cellular processes and components uniquely involved in membrane protein biogenesis and the assembly of these proteins into functional membrane associated units. The proteins which will be initially studied will be two enzymes responsible for phospholipid biosynthesis in Escherichia coli. These two enzymes (phosphatidylserine synthase and phosphatidylglycerophosphate synthase) have been purified to near homogeneity and partially characterized in this laboratory. The first enzyme appears to be a "peripheral" membrane protein while the other enzyme is an "integral" membrane protein. The genes coding for these enzymes have been integrated into multicopy number extrachromosomal vectors which express a level of the respective enzymatic activity in proportion to gene dosage. The process of protein synthesis, processing if it occurs, and final membrane association and assembly will be followed either in vitro using the cloned genes to direct synthesis or in vivo using the cloned genes to highly amplify the gene products. The sequence of events will be determined with respect to time and necessary components in the system. In addition the DNA sequence of the genes will be determined to reveal the protein sequence and the nature of the transcription control regions. The protein sequence will give more information about any processing necessary during assembly. The transcription control regions will be compared with other known sequences. This information will be used to develop models for membrane protein synthesis and assembly.